ABSTRACT
Actin filaments are highly dynamic structures involved in many cellular processes including cell-to-cell/substrate association and cell motility. The actin cytoskeleton is tightly regulated by actin-binding proteins, which include the members of the ADF (actin-depolymerizing factor)/cofilin family. The members of the LIM kinase family of proteins (LIMK1 and 2) regulate actin dynamics by controlling the binding affinity of ADF/cofilin towards actin. LIMK2 has two major splice variants, LMK2a and LIMK2b. We have generated mice lacking LIMK2a expression (LIMK2a KO), to study its specific role in the regulation of the actin cytoskeleton. The LIMK2a KO mice showed a significant prolonged bleeding complication upon injuries compared to wild type mice. This prolonged bleeding prompted us to check the expression of the LIMK2 protein in platelets as it was previously suggested that it is not expressed in platelets. We showed that human and mouse express LIMK2 in platelets and using our LIMK2a KO mice we have identified a potential key role for LIMK2 in platelet functions including platelet spreading, aggregation and thrombus formation.
Subject(s)
Blood Platelets/metabolism , Lim Kinases/metabolism , Platelet Aggregation , Actin Cytoskeleton/metabolism , Animals , Blood Platelets/physiology , Cells, Cultured , Humans , Lim Kinases/genetics , Mice , Mice, Inbred C57BLABSTRACT
Actin is highly abundant in platelets, and its function is dependent on its structure. Actin filaments (F-actin) are dynamic structures involved in many cellular processes including platelet shape changes and adhesion. The actin cytoskeleton is tightly regulated by actin-binding proteins, which include members of the actin depolymerising factor (ADF)/cofilin family. LIM kinase (LIMK) and its phosphatase slingshot (SSH-1L) regulate actin dynamics by controlling the binding affinity of ADF/cofilin towards actin. We hypothesised that the inhibition of LIMK activity may prevent the changes in platelet shape and their function during activation by controlling the dynamics of F-actin. Our results demonstrate that in platelet, inhibition of LIMK by small LIMK inhibitors controls the level of filamentous actin leading to decreased platelet adhesion and aggregation. These findings encourage further studies on controlling platelet function via the cytoskeleton.
Subject(s)
Blood Platelets/metabolism , Lim Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Signal Transduction , Thrombolytic Therapy , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/metabolism , Actins/metabolism , Animals , Blood Platelets/drug effects , Cofilin 1/metabolism , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Down-Regulation/drug effects , Hemorrhage/drug therapy , Humans , Lim Kinases/metabolism , Mice , Phosphoprotein Phosphatases/metabolism , Phosphorylation/drug effects , Platelet Adhesiveness/drug effects , Protein Kinase Inhibitors/therapeutic use , Tail , Thrombosis/drug therapy , rho-Associated Kinases/antagonists & inhibitors , rho-Associated Kinases/metabolismABSTRACT
BACKGROUND: Thrombolytic therapy for acute thrombosis is limited by life-threatening side effects such as major bleeding and neurotoxicity. New treatment options with enhanced fibrinolytic potential are therefore required. Here, we report the development of a new thrombolytic molecule that exploits key features of thrombosis. We designed a recombinant microplasminogen modified to be activated by the prothrombotic serine-protease thrombin (HtPlg), fused to an activation-specific anti-glycoprotein IIb/IIIa single-chain antibody (SCE5), thereby hijacking the coagulation system to initiate thrombolysis. METHODS AND RESULTS: The resulting fusion protein named SCE5-HtPlg shows in vitro targeting towards the highly abundant activated form of the fibrinogen receptor glycoprotein IIb/IIIa expressed on activated human platelets. Following thrombin formation, SCE5-HtPlg is activated to contain active microplasmin. We evaluate the effectiveness of our targeted thrombolytic construct in two models of thromboembolic disease. Administration of SCE5-HtPlg (4 µg/g body weight) resulted in effective thrombolysis 20 minutes after injection in a ferric chloride-induced model of mesenteric thrombosis (48±3% versus 92±5% for saline control, P<0.01) and also reduced emboli formation in a model of pulmonary embolism (P<0.01 versus saline). Furthermore, at these effective therapeutic doses, the SCE5-HtPlg did not prolong bleeding time compared with saline (P=0.99). CONCLUSIONS: Our novel fusion molecule is a potent and effective treatment for thrombosis that enables in vivo thrombolysis without bleeding time prolongation. The activation of this construct by thrombin generated within the clot itself rather than by a plasminogen activator, which needs to be delivered systemically, provides a novel targeted approach to improve thrombolysis.
Subject(s)
Blood Coagulation/drug effects , Fibrinolytic Agents/therapeutic use , Peptide Fragments/biosynthesis , Plasminogen/biosynthesis , Platelet Glycoprotein GPIIb-IIIa Complex/therapeutic use , Single-Chain Antibodies/therapeutic use , Thrombolytic Therapy/methods , Thrombosis/drug therapy , Blood Platelets/drug effects , Blotting, Western , Flow Cytometry , Humans , Peptide Fragments/drug effects , Plasminogen/drug effects , Platelet Activation/drug effects , Platelet Aggregation/drug effects , Platelet Glycoprotein GPIIb-IIIa Complex/immunology , Single-Chain Antibodies/immunology , Thrombosis/bloodABSTRACT
The enzyme-mediated site-specific bioconjugation of a radioactive metal complex to a single-chain antibody using the transpeptidase sortaseâ A is reported. Cage amine sarcophagine ligands that were designed to function as substrates for the sortaseâ A mediated bioconjugation to antibodies were synthesized and enzymatically conjugated to a single-chain variable fragment. The antibody fragment scFv(anti-LIBS) targets ligand-induced binding sites (LIBS) on the glycoprotein receptor GPIIb/IIIa, which is present on activated platelets. The immunoconjugates were radiolabeled with the positron-emitting isotope (64)Cu. The new radiolabeled conjugates were shown to bind selectively to activated platelets. The diagnostic potential of the most promising conjugate was demonstrated in an inâ vivo model of carotid artery thrombosis using positron emission tomography. This approach gives homogeneous products through site-specific enzyme-mediated conjugation and should be broadly applicable to other metal complexes and proteins.
Subject(s)
Aminoacyltransferases/chemistry , Bacterial Proteins/chemistry , Coordination Complexes/chemistry , Copper/chemistry , Cysteine Endopeptidases/chemistry , Single-Chain Antibodies/chemistry , Animals , Mice , Molecular StructureABSTRACT
Lufenuron is an insect growth regulator insecticide mainly used for the control of the cat flea. To understand mechanisms of resistance to lufenuron, we have characterized lufenuron resistance in a natural population of Drosophila melanogaster. In this study we have used precise genetic mapping to identify a mechanism of lufenuron resistance: the overexpression of the cytochrome P450 gene Cyp12a4. Cyp12a4 is predicted to encode a mitochondrial cytochrome P450 enzyme. Expression of Cyp12a4 in D. melanogaster third-instar larvae was detected in the midgut and Malpighian tubules of both lufenuron-resistant and wild-type strains. The level of Cyp12a4 expression in the midgut is higher in the lufenuron-resistant strain than in wild-type strains. Driving the expression of Cyp12a4 in the midgut and Malpighian tubules by using the GAL4/UAS gene expression system results in lufenuron resistance, but it does not result in resistance to three other insecticide classes. Transgenic expression of Cyp12a4 in a ubiquitous expression pattern results in late embryonic lethality, suggesting that high-level ectopic expression of Cyp12a4 is detrimental to development.
